The action of luteinizing hormone release hormone (LHRH) to stimulate luteinizing hormone (LH) secretion by pituitary gonadotrophis is inhibited by testosterone and stimuated by estradiol and progesterone. This steroidal modulation is clearly demonstrable following the administration of physiological concentrations of steriods and LHRH to enzymatically dispersed pituitary cells growth in primary monolayer culture. We have shown that the actions of estradiol and testosterone depend on both steroid interaction with intracellular receptors and protein synthesis. We plan to use this in vitgro model system to expand our studies of the intracellular mechanisms underlying steroid modulation of gonadotroph responsiveness to HRH. In some studies we will utilize cell populations enriched in gonadotrophs, prepared by velocity sedimentation at unit gravity. Initially we will study progesterone enhancement of LHRH action and examine the role of intracellular receptors and protein synthesis in this effect. Next we will characterize those proteins whose rate of synthesis is influenced by testosterone, estradiol and progesterone by monitoring steriod effects on 35S-methionine incorporation into specific proteins in the gonadotroph using two-dimensional polyacrylamide gel electrophoresis and autoradiography. We will also study LHRH effects on these proteins. Further studies will determine whether synthesis of polyadenylated messenger RNA plays a role in steroid effects on LH secretion and protein synthesis. We will also examine the mechanism of LHRH action to stimulate LH secretion in order to identify steroid-sensitive steps in this process. We will characterize steroid effects on LHRH receptors using a whole cell radioreceptor assay; we will examine steroid influences in the stimulation of LH release by cyclic nucleotides and CA++; and we will study steroid effects on LHRH stimulation of protein kinase activity in the gonadotroph, to determine whether steroid modulation of mediated by alterations in intracellular protein phosphorylation.